Enterobacter Amnigenus Research Articles

Phenotypic and Genetic Characterization of Polyhydroxyalkanoate Producing Bacterial Isolates from Hypersaline Water Body, Atlantic Ocean, Nigeria

Introduction: The importance of polyhydroxylalkanoate (PHA) producing bacteria is hinged on the basis that PHA itself possess environmentally advantages over synthetic plastics therefore, the proper identity of these bacteria is inevitable.
 Aim: To determine the bacteria identification methods of phenotypic and genetic identification for marine sourced effective polyhydroxylalkanoate (PHA) production.
 Study Design: Randomised design was employed for PHA producing bacterial isolates.
 Place and Duration of Study: Samples containing PHA-producing bacteria from the hypersaline water body of Nigerian Southern Atlantic Ocean (6.35ᴼ N 3.28ᴼ E -St1; 6.35ᴼ N 3.40ᴼ E -St2; and 6.36ᴼ N 3.47ᴼ E -St3); water depths (0.07m (top), 50m (middle) and 100m (bottom) were collected and investigated between August, 2016-September, 2019.
 Methodology: Bacterial isolates was characterized using standard microbiological and biochemical tests for the phenotypes, and 16S rDNA for the genotypes. Isolates were also screened for PHA potentials using specific primers.
 Results: Genetically identified Bacillus cereus strains LB17 and Bacillus paramycoides SA27 were phenotypically identified as Bacillus badius. Similarly, Acinetobacter calcoaceticus JL11, five strains of Alcaligenes faecalis, seven strains of Bacillus spp., Enterobacter cloacae, Falsochrobactrum ovis, Ochrobactrum ciceri, Providencia stuartii including two species of Pseudomonas and Bordetella trematum were all phenotypically identified as Corynebacterium kutsceri. Invariably, Alcaligenes faecalis (strains PSD10 and DEP8), Pseudomonas aeruginosa (H47921) and Vagococcus fluvialis (AWW1) were identified as Staphylococcus species. All 34 bacterial isolates expressed phaC genes for PHA potential, while Alcaligenes faecalis strains and Bacillus humi (NBPP9) expressed phaC1 for short PHA chain.
 Conclusion: This study established variations in the genetical identity of Corynebacterium kutsceri, Enterobacter amnigenus, Micrococcus luteus, Micrococcus varians, Staphylococcus epidermidis and Staphylococcus saprophyticus when compared with the phenotypic identification methods. But, there was synchronization in the genotypic and phenotypic identity of bacillus species. Records of varietal differences of PHA potential were also obtained from same species in this research. It is therefore pertinent to rely on the genetic identification of bacteria for the effective determination of PHA-producing character as displayed in Bacillus spp.

Exploring the antibacterial potential of plant extracts and essential oils against Bacillus thermophilus in beet sugar for enhanced sucrose retention: a comparative assessment and implications.

Sugar beet is one of the greatest sources for producing sugar worldwide. However, a group of bacteria grows on beets during the storage process, leading to a reduction in sucrose yield. Our study focused on identifying common bacterial species that grow on beets during manufacturing and contribute to sucrose loss. The ultimate goal was to find a potential antibacterial agent from various plant extracts and oils to inhibit the growth of these harmful bacteria and reduce sucrose losses. The screening of bacterial species that grow on beet revealed that a large group of mesophilic bacteria, such as Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas fluorescens, Escherichia coli, Acinetobacter baumannii, Staphylococcus xylosus, Enterobacter amnigenus, and Aeromonas species, in addition to a dominant thermophilic species called Bacillus thermophilus, were found to be present during the manufacturing of beets. The application of 20 plant extracts and 13 different oils indicated that the extracts of Geranium gruinum, Datura stramonium, and Mentha spicata were the best antibacterials to reduce the growth of B. thermophilus with inhibition zones equal to 40, 39, and 35 mm, respectively. In contrast, the best active oils for inhibiting the growth of B. thermophilus were Mentha spicata and Ocimum bacilicum, with an inhibitory effect of 50 and 45 mm, respectively. RAPD-PCR with different primers indicated that treating sugar juice with the most effective oils against bacteria resulted in new recombinant microorganisms, confirming their roles as strong antibacterial products. The characterization of Mentha spicata and Ocimum bacilicum oils using GC/MS analysis identified cis-iso pulegone and hexadecanoic acid as the two main bioactive compounds with potential antibacterial activity. An analysis of five genes using DD-PCR that have been affected due to antibacterial activity from the highly effective oil from Mentha spicata concluded that all belonged to the family of protein defense. Our findings indicate that the application of these pure antibacterial plant extracts and oils would minimize the reduction of sucrose during sugar production.

Pathogenicity test bacteria from Oreochromis niloticus and Clarias gariepinus aquaculture ponds

Present research aimed to examine the pathogenicity opportunities of some bacteria which were isolated from freshwater aquaculture ponds to some fish Oreochromis niloticus, Clarias gariepinus, and Pangasius pangasius. The bacteria were isolated from the water and feces of O. niloticus and C. gariepinus aquaculture in Samarinda, which were identified as Escherichia coli, Enterobacter cloacae, and Enterobacter amnigenus. An 0.1 mL-1 of each bacteria was intramuscular injection and evaluated the clinical signs, eksternal pathology, and fish mortality. The freshwater fish using in this research were Oreochromis niloticus, Clarias gariepinus, and Pangasius pangasius with size 10-15 g from Loa Kulu, Kutai Kartanegara Regency, East Kalimantan, Indonesia. The result showed that each bacteria caused mortality in fish: E. coli bacteria caused fish mortality 23.33-66.67%; E. cloacae 10.00 to 90.00%; and E. amnigenus by 3.33-56.67%. The average death time of E. coli and E. amnigenus in all three fish were under 48 hours, while E. cloacae caused mortality less than 48 hours in C. gariepinus and P. pangasius. In conclusion, E. coli, E. cloacae, and E. amnigenus are putative pathogenic bacterium in O. niloticus, C. gariepinus, and P. pangasius.

Antimicrobial activity of selected plant extracts against common food borne pathogenic bacteria

Increasing antibiotic resistance of important human pathogens calls for investigation and discovery of new antibiotics. Various medicinal plants and spices available in Fiji have antimicrobial potential against various food-borne pathogens. This research aims to address the assumed antimicrobial activity of twelve selected plant extracts against fifteen food-borne pathogens and to evaluate the pathogens resistances. The Kirby-Bauer Method was used in this study to determine the potency of plant extracts. Total Phenolic Content (TPC) was determined by the Folin-Ciocalteu Spectrophotometric method. The chosen plant species samples used were Morinda citrifolia (noni) leaves, Syzygium aromaticum (clove) bud, Psidium guajava (guava) leaves, Ocimum basilicum (basil) leaves, Carica papaya (pawpaw) leaves, Azadirachta indica (neem) seed oil, Citrus limon (lemon) leaves, and Zingiber officinale (ginger) root. Extracts from these eight plants were tested against common food-borne bacteria; Clostridium perfringens, Citrobacter youngae, Enterobacter aerogenes, Enterobacter amnigenus, Enterobacter cloacae, Escherichia coli, Hafnia alveia, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Proteus vulgaris, Proteus mirabilis, Photobacterium damselae, Raoultella ornithinolytica and Vibrio alginolyticus. Both resistance and antibacterial responses were observed for the different plant extracts. All plant extracts were successful in inhibiting bacterial growth for different number of species at varying levels of effectiveness with noni being the most effective. The highest inhibition zones (ZOI>14 mm) were shown by garlic, basil, jasmine and neem. Highest mean ZOI was shown by noni at 11.4 mm followed by garlic at 10.1 mm and clove at 8.4 mm. Minimum inhibitory concentrations were determined for extracts exhibiting antibacterial potential. TPC may attribute to the antibacterial properties of the extracts, however, this needs further investigation. Results indicate presence of possible bioactive compounds in the extracts which can aid in search for new antibiotic drugs from plant sources. Studies that identify the compounds of therapeutically important flora in Fiji is recommended.

Effects of Combined Application of Salicylic Acid and Proline on the Defense Response of Potato Tubers to Newly Emerging Soft Rot Bacteria (Lelliottia amnigena) Infection

Potato soft rot, caused by the pathogenic bacterium Lelliottia amnigena (Enterobacter amnigenus), is a serious and widespread disease affecting global potato production. Both salicylic acid (SA) and proline (Pro) play important roles in enhancing potato tuber resistance to soft rot. However, the combined effects of SA and Pro on defense responses of potato tubers to L. amnigena infection remain unknown. Hence, the combined effects of SA and Pro in controlling newly emerging potato soft rot bacteria were investigated. Sterilized healthy potato tubers were pretreated with 1.5 mM SA and 2.0 mM Pro 24 h before an inoculation of 0.3 mL of L. amnigena suspension (3.69 × 107 CFU mL−1). Rotting was noticed on the surfaces of the hole where the L. amnigena suspension was inoculated. Application of SA and Pro with L. amnigena lowered the activity of pectinase, protease, pectin lyase, and cellulase by 64.3, 77.8, 66.4 and 84.1%, and decreased malondialdehyde and hydrogen peroxide contents by 77.2% and 83.8%, respectively, compared to the control. The activities of NADPH oxidase, superoxide dismutase, peroxide, catalase, polyphenol oxidase, phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-coumaryl-CoA ligase and cinnamate-4-hydroxylase were increased in the potato tubers with combined treatments by 91.4, 92.4, 91.8, 93.5, 94.9, 91.3, 96.2, 94.7 and 97.7%, respectively, compared to untreated stressed tubers. Six defense-related genes, pathogenesis-related protein, tyrosine-protein kinase, Chitinase-like protein, phenylalanine ammonia-lyase, pathogenesis-related homeodomain protein, and serine protease inhibitor, were induced in SA + Pro treatment when compared with individual application of SA or Pro. This study indicates that the combined treatment of 1.5 mM SA and 2.0 mM Pro had a synergistic effect in controlling potato soft rot caused by a newly emerging bacterium.

Complete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium "The Great Five" with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp.

Complete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium "The Great Five" with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp.

Ruta graveolens, Peganum harmala, and Citrullus colocynthis methanolic extracts have in vitro protoscolocidal effects and act against bacteria isolated from echinococcal hydatid cyst fluid.

Echinococcosis is a common and endemic disease that affects both humans and animals. In this study, the in vitro activities of methanolic extracts of Ruta graveolens, Peganum harmala aerial parts, and Citrullus colocynthis seeds against protoscolosis and isolated bacterial strains from hydatid cysts were assessed using disc diffusion methods and Minimum Inhibitory Concentration (MIC). The chemical composition of three methanolic extracts was studied using LC-MS. After 3h of exposure to 40mg/mL R. graveolens extract, a tenfold protoscolocidal effect was seen when compared to the convintional medication (ABZ) for the same duration (P < 0.05). The bacteria listed below were isolated from hydatid cyst fluid collected from a variety of sick locations, including the lung and liver. Micrococcus spp., E. coli, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter amnigenus, Pseudomonas aeruginosa, Staphylococcus xylosus, and Achromobacter xylosoxidans are among the bacteria that have been identified. The most effective extract was R. graveolens, followed by P. harmala and C. colocynthis, according to the results of antibacterial activity using the disc diffusion method. R. graveolens extract had the lowest MIC values (less than 2mg/mL) against all microorganisms tested. This shows that the R. graveolens extract has additional properties, such as the ability to be both scolocidal and bactericidal. Because these bacteria are among the most prevalent pathogenic bacteria that increase the risk of secondary infection during hydatid cysts, the results of inhibitory zones and MICs of the R. graveolens methanol extract are considered highly promising.

Therapeutic Values of Earthworm Species Extract from Azad Kashmir as Anticoagulant, Antibacterial, and Antioxidant Agents.

Aims Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. Methods Ten different earthworms, i.e., Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time. Results Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. Conclusions The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria.

A first report on the identification of a novel archaea, Methanospirillum lacunae from spoilt paints in Lagos, Nigeria using a metagenomic approach

Paint deterioration is a serious esthetic and economic concern attributed to microbial activity.Identifying these microbes is crucial to mitigating their deleterious effects. This paper assessed the bacterial community and population of fresh and spoilt water-based paints from a reputable paint company in Lagos, Nigeria using culture-dependent and metagenomic approaches. Gas Chromatography Mass Spectrophotometry (GCMS) was used to elucidate components of the samples. The bacterial population ranged from 1.4 × 106 to 4.4 × 108 and from 2.3 × 106 to 6.8 × 108 CFU/ml in fresh and spoilt samples respectively. Culture-based technique revealed Bacillus spp. as the dominant bacteria in fresh paint along with Paenibacillus macerans, Corynebacterium striatum, Enterobacter cloacae and Clostridium sphenoides while Pseudomonas spp. were predominant in spoilt paints together with Arthrobacter globiformis, Azotobacter chroccoccum, Burkholderia mallei, Alcaligenes faecalis, Enterobacter amnigenus and Bacillus megaterium. Microbiome sequencing of extracted DNA from spoilt paint revealed a rich bacterial diversity and a surprising archaea population of Methanospirillum lacunae belonging to phylum Euryarchaeota. GC-MS analysis revealed esters as dominant compounds in fresh paints while acids and alcohols suggest that degradation of the components predominate in the spoilt paints. Metagenomic approach thus revealed for the first time, the co-existence of M.lacunae,an archaea with other bacteria in paints.

Distribution and molecular characterization of ESBL, pAmpC β-lactamases, and non-β-lactam encoding genes in Enterobacteriaceae isolated from hospital wastewater in Eastern Cape Province, South Africa.

Globally, there is an increasing occurrence of multidrug-resistant (MDR) Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) β-lactamases in clinical and environmental settings of significant concern. Therefore, we aimed to evaluate the occurrence of ESBL/pAmpC genetic determinants, and some essential non-β-lactam genetic determinants in the MDR phenotypic antimicrobial resistance in Enterobacteriaceae isolates recovered from hospital wastewater. We collected samples from two hospitals in Amathole and Chris Hani District Municipalities in the Eastern Cape Province, South Africa, within October and November 2017. Using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we identified a total of 44 presumptive Enterobacteriaceae isolates. From this, 31 were identified as the targeted Enterobacteriaceae members. Thirty-six percent of these belonged to Klebsiella oxytoca, while 29% were Klebsiella pneumoniae. The other identified isolates included Citrobacter freundii and Escherichia coli (10%), Enterobacter asburiae (6%), Enterobacter amnigenus, Enterobacter hormaechei, and Enterobacter kobei (3%). We established the antibiotic susceptibility profiles of these identified bacterial isolates against a panel of 18 selected antibiotics belonging to 11 classes were established following established guidelines by the Clinical and Laboratory Standard Institute. All the bacterial species exhibited resistance phenotypically against at least four antibiotic classes and were classified as MDR. Notably, all the bacterial species displayed resistance against cefotaxime, ampicillin, nalidixic acid, and trimethoprim-sulfamethoxazole. The generated multiple antibiotic resistance indices ranged between 0.5 to 1.0, with the highest value seen in one K. oxytoca isolated. Molecular characterization via the Polymerase Chain Reaction uncovered various ESBLs, pAmpCs, and other non-β-lactam encoding genes. Of the phenotypically resistant isolates screened for each class of antibiotics, the ESBLs detected were blaCTX-M group (including groups 1, 2, and 9) [51.6% (16/31)], blaTEM [32.3% (10/31)], blaOXA-1-like [19.4% (6/31)], blaSHV [12.9% (4/31)], blaPER [6.5% (2/31)], blaVEB [3.2% (1/31)], blaOXA-48-like and blaVIM [15.4% (2/13)], and blaIMP [7.7% (1/13)]. The pAmpC resistance determinants detected were blaCIT [12.9% (4/31)], blaFOX [9.7% (3/31)], blaEBC [6.5% (2/31)], and blaDHA [3.2% (1/31)]. The frequencies of the non-β-lactam genes detected were catII [79.2% (19/24)], tetA [46.7% (14/30)], sulI and sulII [35.5% (11/31)], tetB [23.3% (7/30)], aadA [12.9% (4/31)], tetC [10% (3/30)], and tetD [3.3% (1/30)]. These results indicate that hospital wastewater is laden with potentially pathogenic MDR Enterobacteriaceae with various antibiotic resistance genes that can be spread to humans throughout the food chain, provided the wastewaters are not properly treated before eventual discharge into the environment.

Patogenesitas Bakteri Enterobacteriaceae pada Ikan Zebra (Danio rerio) Sebagai Hewan Model

Enterobacteriaceae are Gram negative bacteria contain endotoxin and exotoxins which are requirements for pathogenic bacteria and act as opportunistic pathogens. The purpose of this research was to determine the ability of Enterobacteriaceae bacteria to infect zebrafish (Danio rerio) by observing anatomical pathology, mortality, time death and cumulative time of death. The method used in this research is a completely randomized design method (CRD). This research was conducted in three stages, first preparation of zebrasfish as animal model by average size 3-5cm and reach three months old. Second, bacterial cultures from 10 species of Enterobacteriaceae were Escherichia coli 1, E. coli 2, Enterobacter cloacae 1, Acinetobacter calcoaceticus, Enterobacter amnigenus 1, Enterobacter amnigenus 1, Escherichia coli 3, Pantoea spp., E. cloacae 1, E. cloacae 2, E. cloacae 3, cultured in Brain Heart Infused Broth (BHIB) and later media washed with Phospate Buffer Saline (PBS) 0.45%. The third stage was bacterial infection to zebrafish using immersion method with each treatment repeated 3 times and observed for 120 hours. The results showed 10 species that used caused death (mortality), the highest mortality in fish that infected with E. coli 2 and E. cloacae 1 with a percentage of 66.67% infected by E. coli 2 and E. cloacae 1. Anatomical pathology in the external organs and internal organs showed post-infectious symptoms. Infection occurred with mean time to death of 12-86 hours, as long as the cumulative time of fish death caused by E. coli 2, A.cinetobacter calcoaceticus and E. cloacae 1 causing death from 24 hours after infection until the end of observation.

Bacterial microbiota on digestive structure of Cybister lateralimarginalis torquatus (Fischer von Waldheim, 1829) (Dytiscidae: Coleoptera).

In the List of World Edible Insects, Cybister sp. (Dytiscidae) genus of species is known to be consumed by humans. Dried Cybister lateralimarginalis torquatus (Fischer von Waldheim, 1829) which has been collected in Turkey long before and compared to other edible insects having large body, belonging to the Dytiscidae family from the aquatic beetle fauna was aimed to determine microbiota (in digestive structure) of the insect species. In this study, Lelliottia amnigena (Enterobacter amnigenus) (male insect) and Citrobacter freundii (female insect) bacteria species were detected from insect digestion structures. Finally, the DNA sequences of the obtained bacteria were matched from the Gene Bank with the accessory numbers. Moreover, levels of some heavy elements (Al, Cr, Fe, Co, Ni, Cu, Zn, As, Se, Cd, Hg, Pb) were evaluated in this study to observe whether Dytiscidae (Coleoptera) is a useful candidate for biomonitoring studies. The result of the study analyzes agricultural, ecological and health research, influence on the microbial flora and the effect of environment would be and how big the problem we would face in our future. Calculated analysis of the results will give a positive impetus and the fighting method to destroy it in the source.

Assessment and incidence of fish associated bacterial pathogens at hatcheries of Azad Kashmir, Pakistan.

Fish is the most indispensable source of proteins for individuals and have high nutritional value. On the other hand, the fish culturing raised issues of fish health due to close contact between the aquatic environment and the fish pathogens. So, the aim of the current study was to identify the bacterial pathogens and screen the injured Rainbow trout rearing in different trout hatcheries run under fisheries department of the government of Azad Jammu and Kashmir, Pakistan. Seven bacterial pathogens such as Shigella flexneri, Enterobacter amnigenus, Salmonella Typhimurium, Serratia odorifera, Pseudomonas aeruginosa, Streptococcus pyogenes, and Bacillus cereus were isolated and identified. Results revealed that the injury of fish specimens was due to overcrowding. Instead of rainbow coloration, specimens have darker black in color. The water of ponds was not clean and clear and such conditions was because of the greater quantity of feed thrown in the water. It was concluded that poor hygienic water condition and overloading allowed the opportunistic bacterial contaminations to succeed which cause a serious threat to hatcheries.

Simultaneous and sequential influence of metabolite complexes of Lactobacillus rhamnosus and Saccharomyces boulardii and antibiotics against poly-resistant Gram-negative bacteria

For the first time the poly-resistant strains of Gram-negative microorganisms were studied for the sensitivity to combined simultaneous and sequential influence of metabolic complexes of Lactobacillus rhamnosus GG and Saccharomyces boulardii, obtained by the author’s method without using the growth media, with antibiotics. The synergic activity of antibacterial preparations and metabolic complexes of L. rhamnosus GG and S. boulardii were studied using modified disk-diffusive method of Kirby-Bauer. During the sequential method of testing (at first the microorganisms were incubated with structural components and metabolites, then their sensitivity to the antibacterial preparations was determined), we observed increase in the diameters of the zones of growth inhibition of Pseudomonas aeruginosa PR to the typical antibiotics (gentamicin, amіcyl, ciprofloxacin, сefotaxime) and non-typical (lincomycin, levomycetin) depending on the tested combinations. Acinetobacter baumannii PR exhibited lower susceptibility: growth inhibition was seen for the combination with ciprofloxacin, сefotaxime, levomycetin. Susceptibility of Lelliottia amnigena (Enterobacter amnigenus) PR increased to levofloxacin, lincomycin. The zones of growth inhibition of Klebsiella pneumoniae PR increased to gentamicin, amіcyl, tetracycline, сeftriaxone. Maximum efficiency was determined during sequential combination of antibiotics with separate metabolic complexes of L. rhamnosus and S. boulardii, and also their combination (to 15.2, 20.2 and 15.4 mm respectively) compared with their simultaneous use (to 12.2, 15.2 and 13.0 mm respectively) for all the tested poly-resistant pathogens, regardless of the mechanism of action of antibacterial preparation. Metabolic complexes of L. rhamnosus GG and S. boulardii, due to increase in the susceptibility of microorganisms, can decrease the therapeutic concentration of antibiotic, slow the probability of the development of resistance of microorganisms, and are therefore promising candidates for developing “accompanying medications” to antibiotics and antimicrobial preparations of new generation.

The Great Five\u2014an artificial bacterial consortium with antagonistic activity towards Pectobacterium spp. and Dickeya spp.: formulation, shelf life, and the ability to prevent soft rot of potato in storage

“The Great Five” (GF) is an artificial bacterial consortium developed to protect potato tubers from soft rot caused by Pectobacterium spp. and Dickeya spp. To investigate the commercialization potential of the GF, we developed liquid and powder formulations of the consortium and of each of the comprising strains (Serratia plymuthica strain A294, Enterobacter amnigenus strain A167, Rahnella aquatilis strain H145, Serratia rubidaea strain H440, and S. rubidaea strain H469). To form powders, the cells were lyophilized using a newly developed lyoprotectant: Reagent PS. The shelf life of the formulations stored at 8 and 22 °C was monitored for a period of 12 months. The longest shelf life was obtained for formulations stored at 8 °C; however, the viability of all formulations was negatively affected at 22 °C. For the consortium, a 2.5 log10 cfu (colony forming units) drop in cell number was recorded for the liquid formulation after 6 months, while in case of powders, the drop remained below 1 log10 cfu following 12 months. The ability of the powder formulations to preserve biocontrol activity of the consortium was tested on potato tubers treated with the formulations and a mixture of the soft rot pathogens. The inoculated tubers were stored for 6 months at 8 °C to mimic commercial storage conditions. Soft rot severity and incidence on potato tubers treated with formulations were significantly reduced (62–75% and 48–61%, respectively) in comparison to positive control with pathogens alone. The potential use of the newly developed formulations of “The Great Five” for the biocontrol of soft rot is discussed.Key Points• An innovative reagent to protect bacterial cells during lyophilization was developed.• Powder formulations of “The Great Five” prolonged its shelf life.• The powder-formulated “The Great Five” was active against soft rot bacteria on potato tubers.

The Impact of Treated Wastewaters on Fish Bacterial Flora: A Public Health Perspective

Abstract Wastewaters from a treatment plant discharging into a canal harboring fish may present sources of microbiological hazard for wild fish. Such fish, inhabiting microbiologically polluted bodies of water, can be contaminated by human pathogens and, if used for human consumption, may pose a risk to public health. Hence, in this work the aim was to identify tested strains from tissues of wild fish living in the receiving water bodies, captured from locations up to 12 km from the point of discharge of treated water of town Virovitica in order to assess the bacterial threat of the WWTP on fish and potentially on public health. A rather rich diversity of bacterial genera was isolated from gill tissues and internal organs. The most frequent isolate was Aeromonas hydrophila which has gained public health recognition as an opportunistic pathogen. Vibrio cholerae, an indicator bacterium for aquatic contamination, was retrieved from all investigated tissues. Opportunistic human pathogens as well as some zoonotic agents were also retrieved from fish tissues (Enterobacter amnigenus, Acinetobacter spp., Ochrobactrum anthropi, Pseudomonas aeruginosa, Flavimonas (Pseudomonas) oryzihabitans, Shewanella putrefaciens and others). Public health hazard is particularly pronounced regarding local recreational fishermen who fish out, handle and consume fish from respective waters.

Evaluation of anti-microbial activity of filtrates of Lactobacillus rhamnosus and Saccharomyces boulardii against antibiotic-resistant gram-negative bacteria

The article presents the results of the first study on the influence of biologically active substances Lactobacillus rhamnosus GG ATCC 53103 and Saccharomyces boulardii, obtained according to the author`s method, on growth of gram-negative bacteria with broad medical resistance: Pseudomonas aeruginosa PR, Klebsiella pneumoniae PR, Lelliottia amnigena (Enterobacter amnigenus) PR using the spectrophotometric method. Disintegrates of L. rhamnosus GG and S. boulardii were obtained using low-frequency ultrasound processing of suspension of probiotic strains, and metabolites – through cultivation of lactobacteria and saccharomycetes in disintegrates of probiotic microorganisms. To samples of test-cultures with studied filtrates of disintegrates or metabolites we added growth medium and cultivated them (period of monitoring was 5- and 24-hours). Results of the studies were expressed as the percentage of inhibition of increment in polyresistant gram-negative bacteria under the impact of biologically active substances of probiotic microorganisms. Five-hour incubation of test-strains with the studied samples of lactobacteria led to inhibition of their growth properties by 85.6–96.7%. Growth of bacteria under the impact of substances of saccharomycetes was inhibted by 45.1–92.5%. Twenty-four hour exposure of the test-cultures in filtrates of L. rhamnosus GG and S. boulardii caused 100% inhibition of P. aeruginosa and L. amnigena polyresistant strains. Temporal interval of cultivation directly proportionally affected the extent of inhibition of growth of microorganisms: we determined direct correlation dependence within 0.789–0.991. Maximum inhibition of increment of the studied pathogens was observed under the influence of metabolites of lactobacteria, obtained by cultivating primary producers in their disintegrate. We determined a high level of anti-microbial activity of metabolites from L. rhamnosus GG and S. boulardii obtained by cultivation of probiotics in disintegrates against bacteria resistant to a broad range of preparations, which allows us to consider these substances as promising for development of anti-microbial preparations of a new generation against etiologically significant antibiotic-resistant gram-negative microorganisms.

Functional and Genetic Diversity of Bacteria Associated with the Surfaces of Agronomic Plants

The main objective of this study was to evaluate the genetic diversity and agricultural significance of bacterial communities associated with the surfaces of selected agronomic plants (carrot, cabbage and turnip). The bacterial diversity of fresh agricultural produce was targeted to identify beneficial plant microflora or opportunistic human pathogens that may be associated with the surfaces of plants. Bacterial strains were screened in vitro for auxin production, biofilm formation and antibiotic resistance. 16S rRNA gene sequencing confirmed the presence of several bacterial genera including Citrobacter, Pseudomonas, Pantoea, Bacillus, Kluyvera, Lysinibacillus, Acinetobacter, Enterobacter, Serratia, Staphylococcus, Burkholderia, Exiguobacterium, Stenotrophomonas, Arthrobacter and Klebsiella. To address the biosafety issue, the antibiotic susceptibility pattern of strains was determined against different antibiotics. The majority of the strains were resistant to amoxicillin (25 µg) and nalidixic acid (30 µg). Strains were also screened for plant growth-promoting attributes to evaluate their positive interaction with colonized plants. Maximum auxin production was observed with Stenotrophomonas maltophilia MCt-1 (101 µg mL−1) and Bacillus cereus PCt-1 (97 µg mL−1). Arthrobacter nicotianae Lb-41 and Exiguobacterium mexicanum MCb-4 were strong biofilm producers. In conclusion, surfaces of raw vegetables were inhabited by different bacterial genera. Potential human pathogens such as Bacillus cereus, Bacillus anthracis, Enterobacter cloacae, Enterobacter amnigenus and Klebsiella pneumoniae were also isolated, which makes the biosafety of these vegetable a great concern for the local community. Nevertheless, these microbes also harbor beneficial plant growth-promoting traits that indicated their positive interaction with their host plants. In particular, bacterial auxin production may facilitate the growth of agronomic plants under natural conditions. Moreover, biofilm formation may help bacteria to colonize plant surfaces to show positive interactions with host plants.

Effect of disintegrates and metabolites of Lactobacillus rhamnosus and Saccharomyces boulardii on biofilms of antibiotic resistant conditionally pathogenic and pathogenic bacteria

The work presented here is the first to examine the impact of Lactobacillus rhamnosus GG ATCC 53103 and Saccharomyces boulardii metabolites obtained using the author`s method on the formation of biofilm forms of bacteria. The structural components of the probiotic microorganisms were obtained using the method of physical disintegration – low frequency ultrasound waves produced by a G3-109 generator. Metabolites were obtained by cultivating L. rhamnosus and S. boulardii in ultrasound disintegrates of lactobacteria and Saccharomycetes. The impact of biologically active substances on the formation of biofilm of Corynebacterium ulcerans tox+ 112, C. diphtheriae gravis tox+ 108, by antibiotic-resistant Pseudomonas aeruginosa PR, Klebsiella pneumoniae PR, Lelliottia amnigena (Enterobacter amnigenus) PR and P. aeruginosa AТСС 27853 reference strain was studied using the spectrophotometric method. For the first time, we proved that L. rhamnosus GG and S. boulardii metabolites and combinations of metabolites of Saccharomycetes and lactobacteria, obtained by cultivating primary producers in their disintegrates, damage preformed 24-hour biofilms of gram-positive and gram-negative bacteria. The representatives of Corynebacterium exhibited higher sensitivity to the filtrates of disintegrates and products of vital activity of lactobacteria and Saccharomycetes than gram-negative pathogens. High parameters of decrease in optical density of preformed biofilms of Corynebacterium and antibiotic-resistant gram-negative bacteria were observed under the influence of combination of L. rhamnosus GG and S. boulardii metabolites (by 1.3–2.6 times). However, the largest reduction of the optical density of the formed biofilm of all studied strains was observed under the influence of metabolites of lactobacteria (by 1.5–5.3 times). Biologically active substances of L. rhamnosus GG and S. boulardii obtained using the author’s method can be used as candidate preparations which could have a strong influence on the process of the formation of the biofilms and preformed biofilms, and also as a preparations of substitution/addition of therapeutic prescription.

Microbiological assessment of maize ogi cofermented with pigeon pea.

Maize was cofermented with pigeon pea for ogi production and evaluated for microbiological qualities. White maize and pigeon pea were mixed at ratios of 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, with 100:0 serving as the control. Mixtures were cofermented for 96 h at 27 ± 2°C, and microbiological and sensory qualities analyzed were carried out using analysis of variance. Values were significant at p ≤ .05. Results showed that there was a gradual decrease in the pH and increase in total titratable acidity (TTA), respectively, during fermentation in all the samples. At the end of fermentation, pH ranged from 3.47 to 4.27 and TTA ranged from 0.47% to 0.54%, respectively. Total heterotrophic count (THPC) ranged from 5.76 to 5.90 log cfu/g; lactic acid bacteria (LAB) from 6.15 to 5.98 log cfu/g; and yeasts from 5.51 to 5.79 log cfu/g. Microorganisms isolated were lactic acid bacteria (Lactobacillus buchneri, L. casei, L. pentosus, Pediococcus pentosaceus), yeasts (Saccharomyces cerevisiae, Candida kefyr, C. krusei, C. tropicalis), molds (Aspergillus niger, A. flavus, Penicillium oxalicum, Mucor racemosus and Rhizopus stolonifer) and other aerobic bacteria (Klebsiella oxytoca, Enterobacter amnigenus, Staphylococcus xylosus, Bacillus subtilis, B. firmus, Corynebacterium kutscheri, C. striatum, and C. afermentans). In conclusion, the total heterotrophic plate count (THPC) in fortified maize: pigeon pea products was very high. This could constitute health hazards to infants as weaning foods. However, the microbial loads could be reduced through heat treatment as ogi is usually boiled or treated with boiled water before consumption and it can therefore be concluded that the fortified maize: pigeon pea products could be used as weaning foods.